ABSTRACT
4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) is a molecule of physiologically unknown function, although it is predominantly expressed in the brain, spinal cord, liver, and kidney. We identified NIPSNAP1 as a protein that interacts with the neuropeptide nocistatin (NST) from synaptosomal membranes of mouse spinal cord using high-performance affinity latex beads. NST, which is produced from the same precursor protein as an opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ), has opposite effects on pain transmission evoked by N/OFQ. The calculated full-length pre-protein of NIPSNAP1 was 33 kDa, whereas the N-terminal truncated form of NIPSNAP1 (29 kDa) was ubiquitously expressed in the neuronal tissues, especially in synaptic membrane and mitochondria of brain. The 29-kDa NIPSNAP1 was distributed on the cell surface, and NST interacted with the 29-kDa but not the 33-kDa NIPSNAP1. Although intrathecal injection of N/OFQ induced tactile allodynia in both wild-type and NIPSNAP1-deficient mice, the inhibition of N/OFQ-evoked tactile allodynia by NST seen in wild-type mice was completely lacking in the deficient mice. These results suggest that NIPSNAP1 is an interacting molecule of NST and plays a crucial role in pain transmission.
Subject(s)
Analgesics, Opioid/pharmacology , Brain/metabolism , Nerve Tissue Proteins/metabolism , Opioid Peptides/pharmacology , Pain/metabolism , Proteins/metabolism , Spinal Cord/metabolism , Synaptic Transmission/drug effects , Analgesics, Opioid/adverse effects , Animals , Brain/pathology , COS Cells , Chlorocebus aethiops , Humans , Hyperalgesia/chemically induced , Hyperalgesia/genetics , Hyperalgesia/metabolism , Hyperalgesia/pathology , Intercellular Signaling Peptides and Proteins , Membrane Proteins , Mice , Mitochondria/genetics , Mitochondria/metabolism , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/genetics , Opioid Peptides/adverse effects , Pain/genetics , Proteins/agonists , Proteins/genetics , Spinal Cord/pathology , Synaptic Membranes/genetics , Synaptic Membranes/metabolism , Synaptic Transmission/geneticsABSTRACT
We developed novel magnetic nano-carriers around 180 nm in diameter for affinity purification. Prepared magnetic nano-carriers possessed uniform core/shell/shell nano-structure composed of 40 nm magnetite particles/poly(styrene-co-glycidyl methacrylate (GMA))/polyGMA, which was constructed by admicellar polymerization. By utilizing relatively large 40 nm magnetite particles with large magnetization, the magnetic nano-carriers could show good response to permanent magnet. Thanks to uniform polymer shell with high physical/chemical stability, the magnetic nano-carriers could disperse in a wide range of organic solvent without disruption of core/shell structure and could immobilize various kinds of drugs. We examined affinity purification using our prepared magnetic nano-carriers with anti-cancer agent methotrexate (MTX) as ligand. Our magnetic nano-carriers showed higher performance compared to commercially available magnetic beads in terms of purification efficiency of target including extent of non-specific binding protein.
